非特异性过加氧酶（UPO）的研究综述

doi:10.12211/2096-8280.2022-028

摘要/Abstract

摘要：

未活化的C—H键选择性插入活性氧是目前有机合成面临的最具挑战性之一。真菌非特异性过加氧酶（UPO）是一类高度糖基化的硫代血红素酶，催化的反应包括正构烷烃中未活化的C—H键的羟基化、烯烃和芳烃的环氧化、含杂原子（N、S）化合物的氧化、乙醚裂解、N-脱烷基化、脱酰化和酚类的单电子氧化。UPO以H₂O₂为氧供体与电子受体，不需要任何辅因子，是目前最具发展潜力的氧化酶之一。然而，UPO的异源表达困难与选择性差的问题仍限制着UPO的发展。近两年，通过信号肽改造或更换的方法在UPO的异源表达方面取得了重要突破，对UPO结构功能关系的深入研究以及蛋白结构预测算法的发展也将助力UPO的分子改造，为解决UPO选择性差的问题奠定基础。本文聚焦于UPO的异源表达、选择性问题与H₂O₂原位再生，综述了UPO的最新发展以及存在的技术瓶颈，并对解决这些瓶颈问题的方案做出展望。

关键词: 非特异性过加氧酶, 未活化C—H键, 氧化反应, 异源表达, 选择性

Abstract:

The selective insertion of oxygen species through unactivated C—H bonds is one of the most challenging tasks in organic synthesis. Fungal unspecific peroxygenases (UPOs) are a class of highly glycosylated thioheme enzymes that catalyze reactions including hydroxylation of unactivated C—H bonds in n-alkanes, epoxidation of alkenes and aromatics, oxidation of heteroatom (N, S) compounds, ether cleavage, N-dealkylation, deacylation and one-electron oxidation of phenols. As one of the most promising oxidases in synthetic chemistry,UPOs use H₂O₂ as the oxygen donor and the electron acceptor, and do not require cofactors other than heme. This paper reviews the classification and development process of UPOs, and focuses on the heterologous expression, selectivity engineering and H₂O₂in situ regeneration of UPOs. Since the first discovery of UPOs from Agrocybe aegerita in 2004, UPOs have attracted much attention due to the advantages described above. However, the difficulties of heterologous expression and poor selectivity of UPOs still limit their development. The difficulty of heterologous expression makes it hard to mine new variants of UPOs, and native UPOs are difficult to be characterized and applied in biocatalysis due to the slow growth rate of their hosts. In the past two years, important breakthroughs have been made in the heterologous expression of UPOs through the modification or replacement of signal peptides, revealing the important role of signal peptides in this process. However, the specific role of signal peptides in the secretory expression and three-dimensional structure formation of UPOs remain elusive. With the in-depth research on the mechanism of signal peptides affecting the heterologous expression of UPOs and the development of artificial intelligence (AI) algorithms, the combination of genome mining and signal peptide prediction will be the key for discovering new UPOs. The poor selectivity of UPOs also hinders the development and application of UPOs. This paper reviews different types of reactions that UPOs catalyze, and reveals the problem that UPOs have broad substrate range but poor selectivity. In-depth research on the structure-function relationship of UPOs and the development of protein structure prediction algorithms will help the engineering of UPOs and lay a foundation for solving the problem of poor substrate selectivity. This paper also compares several methods for the in situ regeneration of H₂O₂, and concluded that the multi-enzyme cascade method is the most economical and practical method for the in situ regeneration of H₂O₂.

Key words: unspecific peroxygenase(UPO), unactivated C—H bond, oxidation reaction, heterologous expression, selectivity

中图分类号:

Q814

赖铭元, 韦健, 许建和, 郁惠蕾. 非特异性过加氧酶（UPO）的研究综述[J]. 合成生物学, 2022, 3(6): 1235-1249.

Mingyuan LAI, Jian WEI, Jianhe XU, Huilei YU. Review of research on unspecific peroxygenases (UPOs)[J]. Synthetic Biology Journal, 2022, 3(6): 1235-1249.

图/表 16

表1 氧化还原酶分类

Tab. 1 Classification of oxidoreductases (according to the webpage Enzyme Nomenclature of the NC-IUBMB https://iubmb.qmul.ac.uk/enzyme/EC1/)

名称	编号	辅因子	电子受体	催化作用
氧化酶	EC 1.x.3.x	金属离子(Cu等)	O₂	将电子从底物或辅因子转移到分子氧上
氧化酶	EC 1.x.3.x	黄素	O₂、Fe³⁺、醌^-等	将电子从底物或辅因子转移到分子氧上
过氧化物酶	EC 1.11.1.x	血红素	H₂O₂	催化两个电子从底物转移到H₂O₂
过加氧酶	EC 1.11.2.1	血红素	H₂O₂	催化氧原子从H₂O₂转移到底物上
单加氧酶	EC 1.13.12.x	血红素、黄素等	O₂	催化1个氧原子从O₂转移到底物中
双加氧酶	EC 1.13.11.x EC 1.14.11.x EC 1.14.12.x	黄素、金属离子等	O₂	催化2个氧原子从O₂转移到底物中

表2 已成功异源表达的UPO

Tab. 2 UPO that have been successfully heterologously expressed

UPO	来源	异源表达宿主	方法
MroUPO	M. rotula	E. coli	原序列，自诱导培养基^[26]
DcaUPO	D. caldariorum	E. coli	原序列^[27]
CviUPO	C. virescens	E. coli	原序列^[27]
HinUPO	H. insolens	Aspergillus oryzae	原序列^[34]
CciUPO	C. cinerea	Aspergillus oryzae	原序列^[32]
PviUPO	P.virgatula	Aspergillus oryzae	原序列^[34]
ThyUPO	T.hyrcaniae	Aspergillus oryzae	原序列^[34]
LfuUPO	C. fumago	Aspergillus niger	原序列^[39]
AaeUPO (wild-type)	A. aegerita	无细胞	无细胞蛋白表达系统^[38]
AaeUPO (PADAⅠ)	A. aegerita	S. cerevisiae & P. pastoris	信号肽改造^[30-31]
MroUPO	M. rotula	S. cerevisiae & P. pastoris	更换信号肽^[29]
CglUPO	C. globosum	S. cerevisiae & P. pastoris	更换信号肽^[29]
MthUPO	M. thermophila	S. cerevisiae & P. pastoris	更换信号肽^{[29, 36]}
TteUPO	T. terrestris	S. cerevisiae & P. pastoris	更换信号肽^{[29, 36]}
PabUPO	P. aberdarensis	S. Cerevisiae & P. pastoris	更换信号肽^[37]
MfeUPO	M. fergusii	P. pastoris	更换信号肽^[36]
MhiUPO	M. hinnulea	P. pastoris	更换信号肽^[36]
DcaUPO	D. caldariorum	P. pastoris	更换信号肽^[36]
HspUPO	Hypoxylon sp	P. pastoris	原序列^[40]
AniUPO	Aspergillus niger	P. pastoris	信号肽预测^[41]
CabUPO 1	C. aberdarensis	P. pastoris	信号肽预测^[41]
CabUPO 2	C. aberdarensis	P. pastoris	信号肽预测^[41]

图1 硫代血红素酶的氧化机理［细胞色素P450单加氧酶氧化过程包含6个步骤（Step 1-6），且需要NAD（P）H与O2的参与。UPO氧化机理以红色线条表示（Step a-d），UPO不催化分子氧的还原活化，而是直接利用过氧化氢形成具有催化活性的氧-铁基阳离子自由基配合物，因此不依赖于昂贵的NAD（P）H以及拥有更为简洁的电子传递链］

Fig. 1 Mechanism of thioheme-catalyzed oxidation[The process of cytochrome P450 monooxygenase catalyzation consists of six steps and requires the participation of NAD(P)H and O2(Steps 1-6). The catalytic oxidation of UPO is indicated by red lines(Steps a-d). UPO do not catalyze the reduction and activation of molecular oxygen, but directly use hydrogen peroxide to form catalytically active ferric oxide groups, so it does not rely on the expensive NAD(P)H and has a more concise electron transport chain.]

图2 UPO氧化烷烃反应通式

Fig. 2 The general formula for the oxidation of alkanes by UPO

图3 UPO氧化不同烷烃与脂肪酸(a)～(d) AaeUPO氧化直链（支链）烷烃、环烷烃；（e） AaeUPO羟化脂肪酸；（f） MroUPO氧化脂肪酸发生脱羧反应；（g） AaeUPO、CciUPO催化脂肪酸碳链缩短（脱去2个碳原子）

Fig. 3 Oxidation of alkanes and fatty acids by UPO(a)～(d) AaeUPO oxidizes linear (branched) alkanes and cycloalkanes; (e) AaeUPO hydroxylates fatty acids; (f) MroUPO oxidizes fatty acids for decarboxylation; (g) AaeUPO/CciUPO catalyze fatty acid carbon chain shortening (removal of two carbon atom)

图4 UPO氧化烯烃反应通式

Fig. 4 The general formula for the oxidation of olefin by UPO

图5 UPO氧化不同烯烃(a)～(d) AaeUPO氧化不同烯烃，烯烃结构的不同会影响羟化产物与环氧化产物的比例

Fig. 5 Oxidation of Olefins by UPO(a)～(d) AaeUPO oxidizes different olefins, and the difference in olefin structure will affect the ratio of hydroxylation products to epoxidation products

图6 UPO氧化芳烃反应通式

Fig. 6 General formula for the oxidation of aromatic hydrocarbons by UPO

图7 UPO氧化不同芳烃化合物（a） AaeUPO氧化苯［56］；（b） AaeUPO氧化萘［55］；（c） AaeUPO氧化黄酮［57］

Fig. 7 Oxidation of aromatic hydrocarbons by UPO(a) AaeUPO oxidized benzene[56]; (b) AaeUPO oxidized naphthalene[55]; (c) AaeUPO oxidized flavonoids[57]

图8 UPO氧化含S、N或Br原子的化合物

Fig. 8 Oxidation of compounds containing S, N or Br by UPO

图9 AaeUPO氧化芳烃硫醚［58］

Fig. 9 Oxidation of aryl alkyl sulfides by AaeUPO[58]

图10 UPO催化裂解反应（a） AaeUPO催化四氢呋喃发生开环反应；（b） 5-硝基-1，3-苯并二氧戊环（NBD）在UPO作用下脱烷基化生成甲酸和4-硝基邻苯二酚；（c） AaeUPO选择性地催化药物西地那非的N-甲基哌嗪环N-脱烷基化

Fig. 10 UPO catalyzes the cleavage reaction(a) AaeUPO catalyzed the ring-opening reaction of tetrahydrofuran; (b) 5-nitro-1,3-benzodioxole (NBD) was dealkylated under the action of UPO to form formic acid and 4-nitrocatechol; (c) AaeUPO selectively catalyzes the N-dealkylation of the N-methylpiperazine ring of the drug sildenafil

图11 AaeUPO氧化5-羟甲基糠醛（HMF）生成呋喃二羧酸（FDCA）

Fig. 11 Oxidation of 5-hydroxymethylfurfural (HMF) by AaeUPO to 2,5-furandicarboxylic acid (FDCA)

图12 Au-TiO2介导的光催化水氧化反应原位生成H2O2驱动AaeUPO催化氧化反应

Fig. 12 Au-TiO2-mediated photocatalytic water oxidation to generate H2O2in situ for driving AaeUPO-catalyzed oxidation reaction

图13 电化学法原位生成H2O2驱动过加氧酶催化氧化反应的过程

Fig. 13 In situ generation of H2O2 by electrochemical method to drive peroxygenase-catalyzed oxidation

图14 甲酸氧化酶（AoFOx）-过加氧酶偶联系统原位生成H2O2

Fig. 14 In situ generation of H2O2 by a formate oxidase (AoFOx)-peroxygenase coupled system

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